Anti-human Migration Stimulating Factor (MSF) and uses thereof

文档序号：1277033 发布日期：2020-08-25 浏览：21次中文

阅读说明：本技术 抗人迁移刺激因子(msf)及其用途 (Anti-human Migration Stimulating Factor (MSF) and uses thereof ) 是由 A·曼托瓦尼 B·博塔兹 I·拉菲斯 A·英弗扎托 T·古力可于 2018-09-19 设计创作，主要内容包括：本发明涉及能识别和结合包含在人迁移刺激因子(MSF)序列中的表位,且不识别和结合人纤连蛋白1(hFn1)的抗体,以及在诊断方法和治疗中的用途。(The present invention relates to antibodies that recognize and bind to epitopes contained in human Migration Stimulating Factor (MSF) sequences and do not recognize and bind to human fibronectin 1(hFn1), and uses in diagnostic methods and therapy.)

1. An antibody that recognizes and binds to an epitope contained in sequence VSIPPRNLGY (SEQ ID NO:11) of human Migration Stimulating Factor (MSF) but does not recognize and bind to human fibronectin 1(hFn 1).

2. The antibody of claim 1, wherein the epitope consists of the sequence VSIPPRNLGY of human Migration Stimulating Factor (MSF) (SEQ ID NO: 11).

3. Antibody according to claim 1 or 2, characterized in that it is obtained with a peptide of sequence VSIPPRNLGY (SEQ ID NO:11) and/or in that the antibody is selected from the group consisting of IgG, IgM, IgA and IgE antibodies.

4. The antibody of any one of the preceding claims, which is capable of recognizing and binding MSF in an immunoassay, preferably an enzyme-linked immunosorbent assay (ELISA).

5. The antibody of any one of the preceding claims, comprising:

-the complementarity determining region 3 of the heavy chain (CDRH3) having at least 80% identity with the amino acid sequence WDY (SEQ ID NO:12), and/or

The complementarity determining region 3 of the light chain (CDRL3), having at least 80% identity with the amino acid sequence QSYNLVT (SEQ ID NO: 15).

6. The antibody of any one of the preceding claims, comprising:

-the complementarity determining region 3 of the heavy chain (CDRH3) comprising the sequence seq.id No.12, and/or

-complementarity determining region 3 of the light chain (CDRL3) comprising the sequence seq.id No. 15.

7. The antibody of any one of the preceding claims, comprising:

-the complementarity determining region 3 of the heavy chain (CDRH3) having at least 80% identity with the amino acid sequence WDY (SEQ ID NO:12), preferably the CDRH3 comprises SEQ ID NO.12, and/or

-heavy chain complementarity determining region 2(CDRH2) having at least 80% identity with amino acid sequence EIRMKSDNYATYYAESVKG (SEQ ID NO:13), preferably said CDRH2 comprises SEQ ID NO.13, and/or

-heavy chain complementarity determining region 1(CDRH1) having at least 80% identity with the amino acid sequence NDWMN (SEQ ID NO:14), preferably said CDRH1 comprises SEQ ID NO.14, and/or

-the complementarity determining region 3 of the light chain (CDRL3) having at least 80% identity to the amino acid sequence QSYNLVT (SEQ ID NO:15), preferably the CDRL3 comprises SEQ ID NO.15, and/or

-complementarity determining region 2 of the light chain (CDRL2) having at least 80% identity with the amino acid sequence WASTRYS (SEQ ID NO:16), preferably the CDRL2 comprises SEQ ID NO.16, and/or

-complementarity determining region 1 of the light chain (CDRL1) having at least 80% identity with amino acid sequence RSSHYLLNSRTRKNFLS (SEQ ID NO:17), preferably the CDRL1 comprises SEQ ID NO. 17.

8. The antibody of any one of the preceding claims, comprising a heavy chain variable region comprising a sequence having at least 80% identity to the amino acid sequence: EVKIEESGGGLVQPGGSMKLSCVASGFTFSNDWMNWVRQSPEKGLEWV

AEIRMKSDNYATYYAESVKGRFTISRDDSKNSVYLQMNNLRAEDNGIYYCTSWDYWGQGTTLTVSS (SEQ ID NO:18) and/or

A light chain variable region comprising a sequence having at least 80% identity to the amino acid sequence: DIVMSQSPSSLAVSTGEKVTMNCRSSHYLLNSRTRKNFLSWYQQKPG

QSPQLLIYWASTRYSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLHTFGGGTKLEIK(SEQ IDNO:19)。

9. The antibody of any one of the preceding claims, comprising:

-a heavy chain variable region comprising the amino acid sequence:

EVKIEESGGGLVQPGGSMKLSCVASGFTFSNDWMNWVRQSPEKGLEWV

AEIRMKSDNYATYYAESVKGRFTISRDDSKNSVYLQMNNLRAEDNGIYYCTSWDYWGQGTTLTVSS (SEQ ID NO:18) and/or

-a light chain variable region comprising the amino acid sequence:

DIVMSQSPSSLAVSTGEKVTMNCRSSHYLLNSRTRKNFLSWYQQKPG

QSPQLLIYWASTRYSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLHTFGGGTKLEIK(SEQ IDNO:19)，

preferably comprises a heavy chain consisting essentially of the amino acid sequence of SEQ ID No.6 and/or a light chain consisting essentially of the amino acid sequence of SEQ ID No. 8.

10. An antibody capable of recognizing and binding to the epitope recognized by the antibody of the preceding claim, and not recognizing and binding to human fibronectin 1(hFn 1).

11. The antibody of any one of the preceding claims, wherein the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, deimmunized antibodies, fully human antibodies, single chain antibodies, bispecific antibodies, diabodies, scFv, Fab, F (ab)'2 and dimeric, oligomeric or polymeric versions thereof.

12. An in vitro or ex vivo method for the selective detection and/or determination of the protein MSF or fragments thereof, comprising the steps of: detecting in an isolated biological sample obtained from an individual MSF or a fragment thereof, by being able to recognize and bind to an epitope comprised in sequence VSIPPRNLGY (SEQ ID NO:11) of human Migration Stimulating Factor (MSF), but not to recognize and bind to a ligand of human fibronectin 1(hFn1), preferably the ligand is an antibody, more preferably the antibody is an antibody according to any one of claims 1-11.

13. An in vitro or ex vivo method for assessing the risk and/or diagnosis and/or prognosis and/or monitoring the progression and/or monitoring the effect of a therapeutic treatment and/or screening a therapeutic treatment for cancer or an inflammatory condition, preferably asthma or allergy, in an individual comprising the steps of:

a) detecting or determining the amount or activity of the protein MSF or a fragment thereof, or a polynucleotide encoding said protein or fragment thereof, in an isolated biological sample obtained from an individual, and

b) comparison with appropriate controls.

14. The in vitro or ex vivo method of claim 13, wherein the amount of protein MSF or fragment thereof is detected and/or determined by a specific ligand capable of recognizing and binding to an epitope comprised in the human Migration Stimulating Factor (MSF) sequence (VSIPPRNLGY) (SEQ ID NO:11) and not recognizing and binding to human fibronectin 1(hFn 1).

15. The in vitro or ex vivo method according to claim 14, wherein said ligand is an antibody, more preferably said antibody is an antibody according to any one of claims 1-11.

16. The in vitro or ex vivo method of claim 15, comprising the steps of:

a) contacting and incubating the biological sample with the antibody of claims 1-11 such that a MSF-antibody complex is formed as present in MSF;

b) separating the biological sample from the MSF-antibody complex;

c) selectively detecting and/or quantifying MSF bound to the antibody with a detection means for the antibody;

d) comparing the results obtained in c) with control results.

17. The in vitro or ex vivo method of claim 15 or 16, wherein the antibody is immobilized on a solid support, preferably the immobilized antibody is coated on a plate.

18. The in vitro or ex vivo method of claim 16 or 17, wherein the detection means is a detectable antibody, which can be directly detected, optionally wherein the detectable antibody is amplified by a fluorescent reagent, further optionally wherein the detectable antibody is biotinylated, avidin or streptavidin-peroxidase and 3,3',5,5' -tetramethylbenzidine, further optionally wherein the detectable antibody is coupled to peroxidase, 3',5,5' -tetramethylbenzidine, further optionally wherein the detectable antibody is coupled to alkaline phosphatase, p-nitrophenylphosphate and/or 4-methylumbelliferyl phosphate.

19. The in vitro or ex vivo method according to any one of claims 12 to 18, wherein said detection and/or determination of the amount of protein MSF or fragments thereof is performed by an immunoassay, preferably by an ELISA assay.

20. Use of an antibody according to any one of claims 1-11 for detecting and/or quantifying the protein MSF or fragment thereof in an isolated biological sample, preferably wherein detection of MSF or fragment thereof is capable of determining the presence of M2 polarized macrophages and/or M2-like tumor associated macrophages in an individual.

21. A medical use of the antibody of any one of claims 1-11.

22. Molecule capable of modulating MSF expression and/or function, preferably capable of selectively depleting M2 polarized macrophages and/or M2-like tumor associated macrophages, preferably an antibody according to any one of claims 1 to 11, for use in the prevention and/or treatment of a cancer or an inflammatory condition, preferably asthma or allergy.

23.A pharmaceutical composition comprising at least one antibody according to any one of claims 1-11 and a pharmaceutically acceptable excipient, preferably said composition is for parenteral administration, preferably intravenous administration.

24. A nucleic acid molecule encoding the antibody of any one of claims 1-11, preferably comprising a nucleotide sequence consisting essentially of:

GAAGTGAAAATTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACGACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGAATGAAATCTGATAATTATGCAACATATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAATAGTGTCTACCTGCAAATGAACAATTTAAGAGCTGAAGACAATGGCATTTATTACTGTACCAGTTGGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:20) and/or

25. An expression vector encoding the antibody of claims 1-11, preferably comprising the nucleic acid of claim 24.

26. A host cell comprising the nucleic acid of claim 24 or the expression vector of claim 25.

27. Kit for the detection and/or quantification of the protein MSF or fragments thereof in a biological sample, comprising the means for the detection and/or quantification of the antibody and optionally the complex antigen-antibody according to any one of claims 1 to 11.

28. The kit of claim 27, further comprising a solid support to which the antibody is immobilized.

29. The kit of claim 28, wherein the solid support is a microtiter plate.

30. Use of a kit according to claims 27-29 for performing the method according to any one of claims 12-19.

31. The kit of claims 27-29 for use in diagnosing and/or predicting the risk of developing and/or prognosing and/or monitoring progression and/or monitoring the efficacy of a therapeutic treatment and/or screening a therapeutic treatment method in a biological sample of an individual.

32. The kit of claims 30-31, wherein the method is an immunoassay, preferably an ELISA.

33. The kit of claim 30 or 31 or 32, wherein the method detects and/or quantifies human MSF in a biological sample.

Technical Field

The present invention relates to anti-MSF (migration stimulatory factor) antibodies, their medical uses and their use in methods for the treatment or prognosis of inflammatory diseases, particularly tumors.

Prior Art

Plasticity is a hallmark of macrophages: contact with cytokines or microbial products, which acquire specific and distinct phenotypes. M1 and M2 polarized macrophages are extremes of a continuous functional state [1 ]. M1 macrophages mediate resistance to intracellular pathogens and tumors, while M2 polarized macrophages exert immunomodulatory properties and coordinate tissue repair and remodeling. In addition, M2 macrophages also play a role in severe inflammatory diseases, such as asthma and allergic diseases, as well as in resistance to parasites [2 ]. Among cancers, tumor-associated macrophages (TAMs) are the most widespread host inflammatory cells and are an essential component of the tumor microenvironment [3-5 ]. TAMs play a dual role: as expected, on the one hand they elicit an anti-tumor response, but in most cases they coordinate the critical steps of cancer onset and progression [6-8 ]. Upon exposure to tumor-derived products, TAMs acquire an M2-like phenotype, promoting tumor proliferation and progression, angiogenesis and lymphangiogenesis, and inhibiting adaptive immunity. The role of macrophages in tumor growth was demonstrated in experimental models. In humans, CD68+ or CD163+ TAM infiltrating solid tumors, such as breast and pancreatic cancers, are associated with poor outcome [9-11 ]. Similarly, increased TAM numbers are closely associated with shortened progression-free survival in patients with classical hodgkin's lymphoma [12 ]. TAM targeting is a key property of screened anti-cancer compounds, such as trabectedin, an EMA and FDA approved anti-tumor drug [13 ]. Antibodies or single molecules and their precursors that target the macrophage colony stimulating factor (M-CSF or CSF-1) pathway, the primary regulator of macrophage migration, differentiation and survival, are being clinically evaluated, either by themselves or with checkpoint blockade inhibitors [14 ]. Thus, preclinical and clinical data suggest that TAM is a possible biomarker for risk stratification of cancer patients, providing a rationale for targeted treatment of TAM [7,15-17 ]. As mentioned above, there is clearly a need for suitable biomarkers to identify TAMs within tumor mass, as well as to identify M2 polarized macrophages as therapeutic targets. Genotyping of M1 and M2 polarized macrophages and M2-like TAMs revealed a series of M2 polarized cell-selectively expressed genes [18 ]. One of these molecules is a truncated form of human fibronectin 1(hFn1), called Migration Stimulatory Factor (MSF) [18,19] (FIG. 1). The DNA and protein sequences of human Fn1(SEQ ID No:1, NCBI accession AB191261.1, DNA; SEQ ID No:2, NCBI accession BAD52437.1, protein) and MSF (SEQ ID No:3, NCBI accession AJ535086.1, DNA; SEQ ID No:4, NCBI accession CAH60958.1, protein) are as follows. The cDNA for human MSF is identical to the 5 'end of the human fibronectin cDNA up to and including exon III-1a, ending with a unique 195 nucleotide sequence at the 3' end. The expression of MSF is controlled at the transcriptional and post-transcriptional level by a two-step mechanism. The initial MSF transcript was originally produced from the fibronectin gene by read-through of an intron separating exons III-1a and III-1b, followed by intron-endo cleavage to produce a 5.9-kb MSF pre-mRNA (at the transcriptional level). This precursor was further cleaved to form a 2.1-kb mature MSF mRNA containing the 30-bp in-frame coding sequence, continuing to exon III-1a (post-transcriptional level). Mature mRNA is rapidly transported to the cytoplasm where it is translated into the same 70-kDa protein as the N-terminus of full-length fibronectin (up to and including the amino acid sequence encoded by exon III-1 a), adding the MSF-unique (intron-encoded) 10 amino acid long C-terminus (fig. 1). MSF is a carcinoembryonic molecule produced in fetal stages but not normal adult cells. However, different reports describe MSF production by cancer-associated fibroblasts and cancer cells. In this regard, the inventors have recorded MSF production in M2 polarized and tumor associated macrophages (TAMs, which have an M2-like phenotype, promote tumor proliferation and progression, angiogenesis and lymphangiogenesis and inhibit adaptive immunity) [18 ]. WO9000567 relates to migration stimulating factor-1, which is a polypeptide that stimulates migration of normal adult fibroblasts that do not produce the polypeptide per se, has an apparent molecular weight of 70kD as determined by polyacrylamide gel electrophoresis, is cationic at physiological pH, can be precipitated from aqueous solutions with 10% saturated or less ammonium sulfate, is stable in solution at pH2 rather than pH10, is denatured at 56 ℃, is susceptible to trypsin and alkylation/reduction, and binds heparin. Other migration stimulating factors are similar, but anionic. It is produced by embryonic or embryonic-like fibroblasts from cancer patients (but not normal adult skin fibroblasts), and its production can be used as a diagnostic or prognostic indicator for various cancers.

The present inventors' observation of MSF production by M2 polarized macrophages prompted investigation of whether MSF could be used as a diagnostic and prognostic marker. The production of mouse monoclonal antibodies (i.e., mAb7.1) [19,24] raised against a synthetic peptide spanning the C-terminal tail of the aforementioned human MSF (i.e., VSIPPRNLGY [ SEQ ID NO:11 ]). The antibody mAb7.1 was previously reported to recognize rhMSF [19] in a dot blot setup, but there is no data for the use of this antibody in ELISA. In addition, based on existing evidence, the use of this antibody is limited to immunohistochemical procedures [19,23,20 ]. Therefore, there remains a need for antibodies that can detect MSF without binding to fibronectin-1 in different experimental settings.

Summary of The Invention

Thus, the inventors have generated a mouse monoclonal antibody (designated 1G5.3) which selectively recognizes MSF and is effective in quantifying MSF levels in the biological fluids of individuals with pathological conditions associated with macrophage M2 polarization. The antibody was generated by inoculating mice with recombinant human MSF (rhMSF), specifically recognizing the characteristic 10 amino acid long protein C-terminal tail (aa 648-657). Here, the inventors revealed that when comparing the binding of rhMSF and its C-terminus (i.e. the biotinylated synthetic peptide form, biot-VSIPPRNLGY [ SEQ ID NO:11] [ MSF-specific decapeptide (aa.648-657 of SEQ ID NO: 4) containing the biotin moiety biot-Ahx-VSIPPRNLGY linked via the aminocaproic acid (Ahx) arm to the NH2 terminus) as described above to the known antibody mAb7.1 antibody (also called mabVSI7.1, reported in Cancer Res.2005 at 12.1/1; 65(23):10742-9) in a dot blot and ELISA setup, only the antibodies of the invention recognize the biot (Ahx) -VSIPPRNLGY (SEQ ID NO:11) peptide (i.e. the C-terminal specific tail of MSF) and the rhMSF. furthermore, the antibodies of the invention recognize the biot (AhX-VSIPPRNLGY) peptide and the rhMSF. in a dose-dependent manner in conditioned medium as well as purified molecules, the purified antibody of the invention specifically detects rhMSF in dot blot experiment, which expands the application range of the antibody to dot blot. These results indicate that the antibodies of the invention have a unique ability to recognize MSF in different experimental settings, most importantly in ELISA immunoassays for determining MSF levels in biological fluids.

Detailed Description

The authors of the present invention prepared a monoclonal antibody called 1G5.3, obtained by immunizing BALB/c mice with recombinant human MSF (rhMSF). Hybridoma clones secreting the antibody were selected by indirect ELISA for their specificity and selectivity for binding to rhMSF. Antibodies prepared with the selected hybridomas did not actually recognize hFn 1.

Accordingly, an object of the present invention is an antibody that recognizes and binds to an epitope contained in sequence VSIPPRNLGY of human Migration Stimulating Factor (MSF) (aa648 to aa.657[ SEQ ID NO:11] of SEQ ID NO: 4) and does not recognize and bind to human fibronectin 1(hFn 1).

Preferably, the epitope consists of sequence VSIPPRNLGY of human Migration Stimulating Factor (MSF) (aa.648 to aa 657 of SEQ ID No:4 (SEQ ID NO: 11)).

Another object of the invention is an antibody that recognizes and binds human Migration Stimulating Factor (MSF) and does not recognize and bind human fibronectin 1(hFn 1).

Preferably, the antibody of the invention is characterized in that it is obtained using the peptide of sequence VSIPPRNLGY (aa648-657 of SEQ ID No:4 (SEQ ID NO: 11)). In the peptide of sequence VSIPPRNLGY (SEQ ID NO:11) used to obtain the antibody of the present invention, a cysteine is preferably added to the COOH terminus (see SEQ ID NO:9), and more preferably, the peptide is conjugated to Keyhole Limpet Hemocyanin (KLH).

In a preferred embodiment, the antibody of the invention is selected from the group consisting of IgG, IgM, IgA and IgE antibodies.

In a preferred embodiment, the antibodies of the invention are capable of recognizing and binding MSF in an immunoassay, preferably in an enzyme-linked immunosorbent assay (ELISA).

Preferably, the antibody of the invention comprises:

-the complementarity determining region 3 of the heavy chain (CDRH3), having at least 80% identity with the amino acid sequence WDY (SEQ ID NO:12) (aa.120-122 of SEQ ID No. 6), and/or

The complementarity determining region 3 of the light chain (CDRL3), having at least 80% identity with the amino acid sequence QSYNLVT (SEQ ID NO:15) (aa.116-122 of SEQ ID No. 8).

More preferably, it comprises:

-the complementarity determining region 3 of the heavy chain (CDRH3) comprising the sequence seq.id No.12, and/or

-complementarity determining region 3 of the light chain (CDRL3) comprising the sequence seq.id No. 15.

Preferably, the antibody of the invention comprises:

-the complementarity determining region 3 of the heavy chain (CDRH3) having at least 80% identity with the amino acid sequence WDY (SEQ ID NO:12) (aa.120-122 of SEQ ID No. 6), preferably the CDRH3 comprises the sequence of SEQ ID NO:12, and/or

-heavy chain complementarity determining region 2(CDRH2) having at least 80% identity with amino acid sequence EIRMKSDNYATYYAESVKG (SEQ ID NO:13) (aa.69-87 of SEQ ID No. 6), preferably said CDRH2 comprises the sequence of SEQ ID NO:13, and/or

-the complementarity determining region 1 of the heavy chain (CDRH1) having at least 80% identity with the amino acid sequence NDWMN (SEQ ID NO:14) (aa.50-54 of SEQ ID No. 6), preferably said CDRH1 comprises the sequence of SEQ ID NO:14, and/or

-complementarity determining region 3 of the light chain (CDRL3) having at least 80% identity with the amino acid sequence QSYNLVT (SEQ ID NO:15) (aa.116-122 of SEQ ID NO. 8), preferably said CDRL3 comprises the sequence of SEQ ID NO:15, and/or

-complementarity determining region 2 of the light chain (CDRL2) having at least 80% identity with the amino acid sequence WASTRYS (SEQ ID NO:16) (aa.76-82 of SEQ ID NO.8), preferably the CDRL2 comprises SEQ ID NO:16, and/or

-complementarity determining region 1 of the light chain (CDRL1) having at least 80% identity with amino acid sequence RSSHYLLNSRTRKNFLS (SEQ ID NO:17) (aa.44-60 of SEQ ID No. 8), preferably the CDRL1 comprises the sequence of SEQ ID NO: 17.

The Complementarity Determining Region (CDR) peptide is preferably a CDR3 peptide comprising an amino acid sequence selected from the group consisting of: WDY (SEQ ID NO:12) (aa.120-122 of SEQ ID NO: 6) and QSYNLVT (SEQ ID NO:15) (aa.116-122 of SEQ ID NO: 8).

More preferably, the antibody of the invention comprises:

-complementarity determining region 3 of the light chain (CDRL3) having at least 80% identity with the amino acid sequence QSYNLVT (SEQ ID NO:15) (aa.116-122 of SEQ ID No. 8), preferably said CDRL3 comprises the sequence of SEQ ID NO: 15.

Even more preferably, the antibody of the invention comprises:

The antibody of the invention preferably comprises a heavy chain variable region comprising a sequence having at least 80% identity to the following amino acid sequence: EVKIEESGGGLVQPGGSMKLSCVASGFTFSNDWMNWVRQSPEKGLEWVAEIRMKSDNYATYYAESVKGRFTISRDDSKNSVYLQMNNLRAEDNGIYYCTSWDYWGQGTTLTVSS (SEQ ID NO:18) (aa.20-133 of SEQ ID No. 6) and/or

A light chain variable region comprising a sequence having at least 80% identity to the amino acid sequence:

DIVMSQSPSSLAVSTGEKVTMNCRSSHYLLNSRTRKNFLSWYQQKPGQSPQLLIYWASTRYSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLHTFGGGTKLEIK (SEQ ID NO:19) (aa.21-132 of SEQ ID No. 8).

More preferably, the antibody of the invention comprises:

-a heavy chain variable region comprising the amino acid sequence:

EVKIEESGGGLVQPGGSMKLSCVASGFTFSNDWMNWVRQSPEKGLEWVAEIRMKSDNYATYYAESVKGRFTISRDDSKNSVYLQMNNLRAEDNGIYYCTSWDYWGQGTTLTVSS (SEQ ID NO:18) (aa.20-133 of SEQ ID No. 6) and/or

-a light chain variable region comprising the amino acid sequence:

DIVMSQSPSSLAVSTGEKVTMNCRSSHYLLNSRTRKNFLSWYQQKPGQSPQLLIYWASTRYSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLHTFGGGTKLEIK (SEQ ID NO:19) (aa.21-132 of SEQ ID No. 8).

Even more preferably, the antibody of the invention comprises a sequence having at least 80% identity to the amino acid sequence of SEQ ID NO.6 and/or SEQ ID NO. 8. More preferably, the antibody of the invention comprises a heavy chain consisting essentially of the amino acid sequence of SEQ ID No.6 and/or a light chain consisting essentially of the amino acid sequence of SEQ ID No. 8.

It is also an object of the present invention to provide antibodies that recognize and bind to the epitopes recognized by the above antibodies, and that do not recognize and bind to human fibronectin 1(hFn 1).

Preferably, the antibody of the invention is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, deimmunized antibodies, fully human antibodies, single chain antibodies, bispecific antibodies, diabodies, scFv, Fab, F (ab)'2 and dimeric, oligomeric or polymeric versions thereof.

Yet another object of the present invention is an in vitro or ex vivo method for selectively detecting and/or determining the amount of protein MSF or fragments thereof, comprising the steps of: MSF or a fragment thereof is detected in an isolated biological sample obtained from an individual by being able to recognize and bind to an epitope comprised in sequence VSIPPRNLGY (SEQ ID NO:11) (aa.648 to aa 657 of SEQ ID No. 4) of human Migration Stimulating Factor (MSF), but not to recognize and bind to a specific ligand of human fibronectin 1(hFn1), preferably the ligand is an antibody, more preferably the antibody is an antibody as described above.

Another object of the invention is an in vitro or ex vivo method for assessing the risk and/or diagnosing and/or prognosing and/or monitoring the progression and/or monitoring the efficacy of a therapeutic treatment and/or screening a therapeutic treatment for cancer or an inflammatory condition, preferably asthma or allergy, in an individual, comprising the steps of:

b) comparison with appropriate controls.

Preferably in the in vitro or ex vivo method of assessing the risk of cancer or inflammatory pathology and/or diagnosis and/or prognosis and/or monitoring the progress and/or monitoring the efficacy of a therapeutic treatment and/or screening a therapeutic treatment as defined above, the amount of the protein MSF or a fragment thereof is detected and/or determined by a specific ligand capable of recognizing and binding to an epitope comprised in the sequence VSIPPRNLGY (SEQ ID NO:11), aa.648 to aa.657 of SEQ ID No.4 from human Migration Stimulating Factor (MSF) and not recognizing and binding to human fibronectin 1(hFn1), preferably the ligand is an antibody, more preferably the antibody is as defined above.

Said detection and/or determination of the protein MSF or fragments thereof is preferably carried out by means of an immunoassay, preferably an ELISA assay.

The in vitro or ex vivo method of the invention preferably comprises the steps of:

a) contacting and incubating the biological sample with the antibody such that a MSF-antibody complex is formed as in the presence of MSF;

b) separating the biological sample from the MSF-antibody complex;

c) selectively detecting and/or quantifying MSF bound to the antibody with a detection means for the antibody;

d) comparing the results obtained in c) with control results.

Preferably the antibody is immobilized on a solid support, preferably the immobilized antibody is coated on a plate, preferably a microtiter plate.

The detection means is preferably a detectable antibody. The detectable antibody is preferably directly detectable, optionally the detectable antibody is amplified by a fluorescent agent, optionally the detectable antibody is biotinylated, the detecting means is avidin or streptavidin-peroxidase and 3,3',5,5' -tetramethylbenzidine, or the detectable antibody is conjugated to alkaline phosphatase, the detecting means is p-nitrophenyl phosphate and/or 4-methylumbelliferyl phosphate.

Said detection and/or determination of the amount of the protein MSF or fragment thereof is preferably performed by an ELISA assay.

Another object of the invention is the use of the above-described antibody for detecting and/or quantifying the protein MSF or a fragment thereof in an isolated biological sample, preferably wherein the detection of MSF or a fragment thereof is capable of determining the presence of M2 polarized macrophages and/or M2-like tumor associated macrophages in an individual.

Still another object of the present invention is a medical use of the above antibody.

Another object of the present invention is a molecule capable of modulating MSF expression and/or function, preferably capable of selectively depleting M2 polarized macrophages and/or M2-like tumor associated macrophages, preferably an antibody as described above, for use in the prevention and/or treatment of a cancer or inflammatory condition, preferably asthma or allergy.

Another object of the present invention is a pharmaceutical composition comprising at least one of the above-mentioned antibodies and a pharmaceutically acceptable excipient, preferably said composition is for parenteral administration, preferably intravenous use.

Further object of the present invention are nucleic acid molecules encoding the above-mentioned antibodies, preferably comprising a nucleotide sequence consisting essentially of:

GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAACAGGAGAGAAGGTCACTATGAACTGCAGATCCAGTCACTATCTGCTCAACAGTAGAACCCGAAAGAACTTCTTGTCTTGGTACCAACAGAAACCAGGACAGTCTCCTCAACTGCTGATCTACTGGGCATCCACTAGGTATTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAACAATCTTATAATCTTCACACGTTCGGAGGGGGGACCAAGTTGGAAATAAAG (nt.61-396 of SEQ ID No. 7) (SEQ ID NO:21), an expression vector encoding the above antibody, preferably a host cell comprising said nucleic acid or comprising said nucleic acid, or said expression vector.

Even more preferably, the nucleic acid sequences of the invention have at least 80% identity with the sequences of SEQ ID NO:20 and/or SEQ ID NO: 21. More preferably, the nucleic acid sequence of the invention consists essentially of SEQ ID No.20 and/or SEQ ID No. 21. Even more preferably, the nucleic acid of the invention consists essentially of SEQ ID No.5 and/or SEQ ID No. 7.

Another object of the present invention is a kit for the detection and/or quantification of the protein MSF or its fragments in a biological sample, comprising the above-mentioned means for the detection and/or quantification of the antibody and optionally of the complex antigen-antibody, and the use of said kit for carrying out the above-mentioned method.

Preferably the kit further comprises a solid support for the immobilization of the antibody, said solid support preferably being a microtiter plate.

Another object of the invention is the use of the above-described kit for carrying out the above-described method and the use of the above-described kit for diagnosing and/or predicting the risk of developing and/or prognosis and/or monitoring the progression and/or monitoring the efficacy of a therapeutic treatment and/or screening a therapeutic treatment method in a biological sample of an individual.

Preferably the above method is an immunoassay, preferably an ELISA, and/or it detects and/or quantifies human MSF in a biological sample.

In the above method, an amount or activity of the protein MSF or fragment thereof, or the polynucleotide or fragment thereof, in the isolated biological sample obtained from the individual that is greater than a control amount or activity indicates that the individual is at increased risk of developing or affected by the disease.

Preferably the biological sample is isolated from a human individual, optionally wherein the human individual is a cancer patient or a patient with an inflammatory condition, and the measuring step a) further comprises comparison with a standard curve to determine the level of MSF compared to normal individuals. Preferably the biological sample is a tumor lysate, plasma, serum or urine.

In vitro methods of monitoring progression and/or monitoring efficacy of a disease treatment as described above, preferably comprise the steps of:

a) determining in an isolated biological sample obtained from the individual a change in the amount or activity of the protein MSF or fragment thereof, or a polynucleotide encoding said protein or fragment thereof, and

b) comparing the change determined in step a) with a suitable control change.

Preferably the in vitro method for assessing the risk and/or diagnosis and/or prognosis of cancer or an inflammatory condition, preferably asthma or allergy as described above, comprises the steps of:

a) determining the amount or activity of the protein MSF or fragment thereof, or of a polynucleotide encoding said protein or fragment thereof, in an isolated biological sample obtained from an individual, and

b) comparing the amount or activity determined in step a) with a suitable control amount or activity,

wherein an amount or activity of the protein MSF or fragment thereof, or the polynucleotide or fragment thereof, in an isolated biological sample obtained from the individual that is greater than a control amount or activity indicates that the individual is at increased risk of developing or is affected by cancer or an inflammatory condition.

Preferably the in vitro method of monitoring progression and/or monitoring efficacy of a therapeutic treatment of cancer or an inflammatory condition is as described above, comprising the steps of:

b) comparing the change determined in step a) with a suitable control change.

In a preferred aspect of the invention, step a) of the above method is carried out by a method for detecting and/or determining the amount of MSF protein or fragments thereof as described above. The antibodies of the invention can be used to purify culture supernatants by immunoaffinity chromatography (IAC) as described above, e.g., secreted rhMSF in cell lines transfected with DNA sequences encoding rhMSF, and/or native (non-recombinant) MSF protein expressed and secreted in vivo and in vitro, e.g., M2 macrophages, cancer embryonic fibroblasts, and cancer cells. The antibodies of the invention may also be used in immunohistochemistry to recognize MSF in human tissues under physiological conditions and pathological conditions primarily associated with cancer.

In preferred embodiments, the amount of MSF in the isolated biological sample obtained from the individual is higher than the control, which indicates that the individual has, or is at increased risk of developing, a cancer or inflammatory condition.

In other embodiments of the methods of the invention, the amount of MSF in the isolated biological sample obtained from the individual is lower than the control amount, indicating that the individual is progressing towards an improvement in the condition. In the present invention, suitable controls may be selected from among measurements in healthy patients, patients with a non-inflammatory condition or unaffected by cancer, patients affected by an inflammatory or cancer condition prior to a therapeutic treatment, patients affected by an inflammatory condition or cancer during a therapeutic treatment, patients with an inflammatory condition or cancer at different time points in the disease process. The isolated biological sample of the above method is preferably plasma, blood, serum, tissue obtained by surgical resection, tissue obtained by biopsy, cell culture, cell supernatant, cell lysate, tissue sample, organ, bone marrow. Another object of the invention is a method of treating and/or preventing cancer or other inflammatory diseases comprising administering to an individual an antibody of the invention. Another object of the invention is an expression vector encoding the above antibody, preferably comprising the above nucleic acid. Another object of the present invention is a host cell comprising the above-mentioned nucleic acid, or the above-mentioned vector, and a method for producing the above-mentioned antibody, comprising the steps of culturing the above-mentioned host cell and purifying the antibody from the cell culture. In other embodiments of the invention, a device or kit for analyzing a patient sample is provided. Alternatively, the reagents may be provided in a kit comprising the reagents in suspended (suspension) or suspendable (suspendable) form, e.g., reagents bound to beads suitable for flow cytometry, preferably magnetic beads coated with antibody capture, or custom dried antibody mixtures for multicolor analysis and/or columns with filter cartridges of certain size and/or associated Specific Antibody Filters (SAF), etc. The instructions may include instructions for performing an antibody-based flow cytometry assay. The detection means is preferably a means capable of detecting and/or measuring the amount of MSF, e.g. a means capable of detecting complex antigen-antibody, such as an enzyme-coupled secondary antibody, a luminescent substrate, antibody capture coated magnetic beads, a custom dried antibody mix and/or a column with a filter cartridge of a certain size and/or a combined Specific Antibody Filter (SAF). Preferably the kit further comprises a solid support, wherein the antibody is immobilized. Preferably, the kit of the present invention is an immunoassay kit, more preferably an ELISA kit. The means for detecting and/or quantifying the antigen-antibody complex may be a means for detecting and/or quantifying the catalytic activity of MSF, such as a secondary antibody coupled to an enzyme, a luminescent substrate. These means are also well known in the art. The kit according to the invention may also comprise typical auxiliaries, such as buffers, carriers, dyes, etc. and/or instructions for use. The kit may further comprise control means for comparing the increase in the amount of MSF to a suitable control value. For example, reference to known standards, control values can be obtained from normal individuals or normal populations. In the present invention, a "control result" may be a result obtained from a sample isolated from a healthy individual, or from a patient suffering from a disease other than cancer or an inflammatory disease.

For methods of monitoring the progression of cancer and inflammatory disease, the control results can be the results of samples isolated from the same individual at different time points prior to initiation of treatment, at different time points during treatment, and the like.

In the method of monitoring the efficacy of a treatment, the control sample may be a sample taken from the same individual prior to initiation of treatment or at a different time during treatment. By "monitoring efficacy" is meant monitoring the pharmacological profile of the drug. In the method of screening for treatment of cancer and inflammatory diseases, the control sample may be a sample taken from an untreated individual or an individual treated with a test substance, or an individual treated with a reference treatment. Reference treatments for particular cancer and inflammatory diseases are known to those skilled in the art. "screening" refers to assessing whether a drug is biologically or pharmacologically active against cancer and inflammatory diseases. In the present invention, the expression "detecting" refers to any use of any method of observing, assessing or quantifying the presence of an antibody in a sample or the absolute or relative amount of said antibody in a sample, e.g. by chemiluminescence, fluorescence, spectrophotometry, etc. In the present invention, the expression "quantifying" may be understood as determining the amount, concentration or level of MSF or related antibodies, preferably by semi-quantitative or quantitative methods. The term "amount" as used in the specification means, but is not limited to, an absolute or relative amount of a protein, and any other value or parameter that is related to or derivable from the latter. Such values or parameters include signal intensity values obtained from physical or chemical properties of the protein, signal intensity values obtained by direct measurement of intensity values in, for example, immunoassays, mass spectrometry, or nuclear magnetic resonance. In addition, these values or parameters include those obtained by indirect measurements.

The above antibodies include human and animal monoclonal antibodies or fragments thereof, single chain antibodies and fragments thereof and miniantibodies, bispecific antibodies, diabodies, triabodies, or dimeric, oligomeric or polymeric versions thereof. Also included are peptidomimetics or peptides derived from the antibodies of the invention, e.g., which include one or more CDR regions, preferably the CDR3 region. Also included are human monoclonal antibodies and peptide sequences, based on structural active linkages, generated by a manual modeling process (greenj. et al, j.med. chem.,1994, volume 37, page 1035-. Preferably the antibody is selected from the group consisting of: intact immunoglobulin (or antibody), Fv, scFv (single chain Fv fragment), Fab, F (ab)'₂An antibody-like domain, an antibody-mimicking domain, a single antibody domain, a multimeric antibody, a peptide or a proteolytic fragment comprising an epitope-binding region. The term "antibody" in the present invention is used in the broadest sense and includes a variety of antibodies and antibody mimetic structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, deimmunized antibodies, chimeric antibodies, nanobodies, antibody derivatives, antibody fragments, anticalin, designed ankyrin repeats (DARPins), affibodies, affilins, adhesins (affimers), affitines, alpha bodies (alphabodies), avimers, fynomers, minibodies (minibodies), and other binding domains, provided that they exhibit desirable binding activity to an antigen. Antigen-binding fragments of these antibodies can be produced using conventional techniques. Examples of such fragments include, but are not limited to, Fab, F (ab')₂And Fv fragments. Antibody fragments and derivatives produced using genetic engineering techniques are also contemplated. Unless otherwise indicated, the terms "antibody" and "monoclonal antibody" include intact antibodiesBodies and antigen-binding fragments thereof. An "antibody fragment" refers to a molecule other than an intact antibody, which molecule comprises a portion of an intact antibody that binds to an antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and antibody fragments. The Fv of VH and VL are also known as "nanobodies". The term "mimobody" refers to an organic compound or binding domain that is not a derivative of an antibody but specifically binds to an antigen in the same manner as an antibody. They include antiporters, designed ankyrin repeats, affibodies, affilins, avidin, affitine, alpha bodies, avimers, fynomers, minibodies, and the like. The term "chimeric" antibody refers to an antibody in which the heavy and/or light chain portions are derived from one particular source or species, while the remaining heavy and/or light chains are derived from a different source or species. The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as described herein. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody-encoding sequence. The definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. In humans, the antibody isotypes are IgA, IgD, IgE, IgG and IgM. An antibody is "humanized" to refer to a chimeric antibody comprising the amino acid residues of the non-human hypervariable region (HVR) and the amino acid sequence from the remaining human region (FR: framework region). In some embodiments, a humanized antibody can comprise substantially at least one entire variable region, typically two, in which all or substantially all of the HVRs (e.g., CDRs) correspond to a relative portion of a non-human antibody and all or substantially all of the FRs correspond to a relative portion of a human antibody. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies, e.g., non-human antibodies, refer to antibodies that have been humanized. "deimmunized" antibodies are the basic requirement for T cell stimulation by disrupting HLA binding-an antibody that reduces immunogenicity. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody comprising the population is identical and/or binds the same epitope, except for possible antibody variants, e.g., comprising naturally occurring mutations or produced during the preparation of the monoclonal antibody, which variants are typically present in lower amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA-based methods, phage display methods, and methods using transgenic animals containing all or part of a human immunoglobulin locus. In the context of the present invention, "an antibody that binds MSF but does not recognize Fn 1" includes modifications to the antibody of the present invention that maintain the above-mentioned specificity. Such changes include, for example, conjugation to effector molecules such as chemotherapeutic or cytotoxic agents and/or detectable reporter moieties.

Bispecific antibodies are macromolecular heterobifunctional cross-linkers that have two different binding specificities within one molecule. Within this group, for example, bispecific (bs) IgG, bs IgM-IgA, bs IgA-dimer, bs (Fab')₂,bs(scFv)₂Diabodies and bs-bis-Fab Fc (Cao y. and suresh m.r., Bioconjugate chem.,1998, vol 9, p 635-644).

Peptide mimetics refers to low molecular weight peptide components, which means structures that mimic the native peptide component, or a template that induces the formation of specific structures in adjacent peptide sequences (Kemp DS, Trends biotechnol.,1990, page 249-255). The peptidomimetic may be, for example, derived from a CD3 domain. The methodological mutational analysis of a given peptide sequence, i.e. by alanine or glutamate scanning mutagenesis analysis, allows the identification of peptide residues critical for procoagulant activity. Another possibility to improve the activity of certain peptide sequences is the use of peptide libraries in conjunction with high throughput screening.

The term antibody may also include substances obtained by analysis of data relating to structure-activity relationships. These compounds are also useful as peptidomimetics (Grassy G. et al, Nature Biotechnol.,1998, Vol.16, p. 748-752; Greer J. et al, J. Med. chem.,1994, Vol.37, p. 1035-1054).

The term antibody may also include proteins produced by expression of altered immunoglobulin-encoding regions in a host cell, e.g., "technically modified antibodies" such as synthetic antibodies, chimeric or humanized antibodies or mixtures thereof, or antibody fragments which partially or completely lack constant regions, e.g., Fv, Fab' or F (ab)2, and the like. In these techniques modified antibodies, for example, one or more portions of the light and/or heavy chains may be substituted. These molecules may, for example, comprise antibodies consisting of a humanized heavy chain and an unmodified light chain (or chimeric light chain), or vice versa. The term Fv, Fc, Fd, Fab, Fab 'or F (ab)'₂As described in the prior art (Harlow E. and Lane D, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

The invention also encompasses the use of Fab fragments or F (ab)'₂A fragment derived from a monoclonal antibody (mAb) directed against MSF. Preferably, the hetero-framework and constant regions are selected from the group consisting of human immunoglobulin classes and isotypes, such as IgG (subtypes 1-4), IgM, IgA and IgE. During the course of an immune response, a type switch of immunoglobulins may occur, for example a switch from IgM to IgG; where the constant region is switched from, for example, y. Type conversion may also occur in a targeted manner by genetic engineering methods ("targeted type conversion recombination"), as is known in the art (Esser c. and radbreak a., annu. rev. immunol.,1990, volume 8, page 717-. However, the antibodies of the invention need not exclusively include human sequences of immunoglobulins.

In one embodiment, the humanized antibody comprises Complementarity Determining Regions (CDRs) from a murine monoclonal antibody inserted within selected human antibody sequence framework regions. However, human CDR regions may also be used. Preferably, the variable regions in the human light and heavy chains are technically altered by one or more CDR exchanges. It is also possible to use all 6 CDRs or different combinations of less than 6 CDRs. The term antibody may also include substances obtained by analysis of data relating to structure-activity relationships. These compounds are also useful as peptidomimetics (Grassy G. et al, Nature Biotechnol.,1998, Vol.16, p. 748-752; Greer J. et al, J. Med. chem.,1994, Vol.37, p. 1035-1054).

Antibodies of the invention also include those antibodies whose binding characteristics have been improved by direct mutation, affinity maturation, phage display. Affinity or specificity may be altered or improved by mutations in any CDR of an antibody of the invention. The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in binding of the antibody to an antigen. The variable domains (or regions) of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, each domain comprising 4 framework conserved regions (FRs) and three hypervariable regions (HVRs see, e.g., Kindt et al, "Kuby Immunology", 6 th edition, w.h.freeman and co., page 91, 2007). A single V_HOr V_LThe domain may be sufficient to confer antigen binding specificity. In addition, libraries of complementary VL or VH domains can be screened by isolating antibodies that bind to a particular antigen using VH or VL domains from antibodies that bind to the antigen (see, e.g., Portolano et al, J.Immunol.150:880-887, 1993; Clarkson et al, Nature352:624-628, 1991).

In another aspect, an antibody or derivative thereof comprises a heavy chain (VH) variable region sequence and/or a light chain (VL) variable region sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the CDRs comprised in each of the amino acid sequences of SEQ ID NO:6 or SEQ ID NO:8 as described above. In some embodiments, the VH sequence and/or VL sequence is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to aa 20-133 of SEQ ID NO:6 or aa.21-132 of SEQ ID NO:8, respectively, and comprises a substitution (e.g., a conservative substitution), insertion, or deletion compared to a reference sequence, while an anti-MSF antibody comprising the sequence maintains the ability to bind to an epitope. Antibody-like domains include binding proteins that are structurally related to antibodies, such as T cell receptors. The antibodies of the invention also include functional equivalents, which include polypeptides having amino acid sequences substantially identical to the amino acid sequences of the variable or hypervariable regions of the antibodies of the invention.

In the present invention, "at least 80% identity" means that identity may be at least 80%, 87% or 90% or 95% or 100% sequence identity to the referenced sequence. This applies to all the mentioned% identities. Preferably,% identity relates to the full length of the mentioned sequences.

The sequences of the invention include amino acid sequences having at least 70, preferably at least 80, more preferably at least 90% identity or homology to the sequence. "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve full-length maximum alignment of the compared sequences. The antibody of the present invention may have, for example, the following dissociation constant (K)_D)：<100nM,<10nM,<1nM,<0.1nM,<0.01nM, or<0.001nM or less, e.g. from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13And M. Recombinant and/or biotechnological derivatives as well as fragments of the above mentioned antibodies are also encompassed by the present invention, provided that the binding activity of the antibody and its functional specificity are maintained. The above antibodies are used as a medicament, preferably for the treatment and/or prevention of cancer or an inflammatory condition. Preferred diseases that can be treated with the antibodies of the invention are neoplastic diseases, such as solid tumors (e.g. breast cancer, lung cancer, colorectal cancer, pancreatic cancer such as Pancreatic Ductal Adenocarcinoma (PDAC), glioma, prostate cancer, thyroid cancer, ovarian cancer, liver cancer, neuroblastoma, melanoma) and non-solid tumors (e.g. leukemia, melanoma, prostate cancer,multiple myeloma and lymphoma, chronic myeloproliferative neoplasm).

In the context of the present invention, in addition to cancer, inflammatory conditions include asthma, allergy, sepsis and infectious diseases; autoimmune diseases, such as rheumatoid arthritis and autoinflammatory diseases, such as inflammatory bowel disease; chronic degenerative diseases, involving tissue remodeling and fibrosis, such as idiopathic pulmonary fibrosis, cirrhosis and chronic heart failure.

In the context of the present invention, "cancer" or "tumor" includes primary and metastatic tumors, as well as refractory tumors, tumors expressing MSF, solid or non-solid tumors. Examples of solid tumors are: breast cancer, lung cancer, colorectal cancer, pancreatic cancer such as pancreatic ductal adenocarcinoma, glioma, lymphoma, prostate cancer, thyroid cancer, ovarian cancer, liver cancer, neuroblastoma, melanoma. Examples of non-solid tumors are: leukemia, multiple myeloma and lymphoma, chronic myeloproliferative neoplasm.

A further aspect of the invention is a nucleic acid encoding or hybridizing to the above-mentioned antibody or consisting of a corresponding degenerate sequence. The scope of the present invention includes expression vectors encoding the above-described antibodies, preferably comprising the above-described nucleic acids. Also within the scope of the invention are host cells comprising the above-described nucleic acids or the above-described vectors. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells," which include transformed primary cells and progeny derived therefrom, not counting the number of steps. The nucleic acid content of the progeny is not necessarily identical to that of the parent cell and may comprise mutations. In the present invention, mutant progeny are included which have the same function or biological activity as screened or selected in the originally transformed cell. The nucleic acids of the invention may be used to transform a suitable mammalian host cell. Mammalian cells useful as expression hosts are well known and include, for example, CHO and BHK cells. Prokaryotic hosts include, for example, E.coli, Pseudomonas, Bacillus, and the like. The antibodies of the invention may be fused to additional amino acid residues, such as a tag to facilitate isolation. The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors in the form of self-replicating nucleic acid structures, as well as vectors that enter the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. These vectors are currently referred to as "expression vectors". Any suitable expression vector may be used, for example, a prokaryotic cloning vector such as a plasmid for E.coli, e.g., colE1, pCR1, pBR322, pMB9, pUC. Expression vectors suitable for expression in mammalian cells include derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences. The expression vector used in the present invention comprises at least one expression control sequence operably linked to a DNA sequence or fragment which must be expressed. Still another object of the present invention is a method for the treatment and/or prevention of angiogenesis-related diseases, preferably neoplastic conditions such as melanoma, glioblastoma, colorectal cancer, breast cancer, renal cancer, ovarian cancer, cervical cancer, non-small cell lung cancer and/or metastases, retinopathies such as diabetic retinopathy, age-related exudative macular degeneration neuroangiogenesis, macular edema caused by retinal artery occlusion, choroidal neuroangiogenesis resulting from pathological myopia, comprising administering to a patient in need thereof a therapeutically effective amount of the above antibody or synthetic or recombinant fragment thereof. Another object of the present invention is a pharmaceutical composition comprising at least the above-mentioned antibody or a synthetic or recombinant fragment thereof, and a pharmaceutically acceptable excipient, preferably said composition is for parenteral administration, preferably intravenous use. The compositions comprise an effective amount of an antibody and/or recombinant or synthetic antigen-binding fragments thereof. Pharmaceutical compositions are conventional in the art and can be prepared by those skilled in the art based on conventional general knowledge. Formulations for use in the treatments described herein may, for example, comprise the above-described antibody at a concentration of from about 0.1mg/ml to about 100mg/ml, preferably from 0.1 to 10mg/ml, more preferably from 0.1 to 5 mg/ml. In other formulations, the antibody concentration may be lower, for example at least 100 pg/ml. The antibodies of the invention are administered to a patient in one or more treatments. Depending on the type and severity of the disease, a dosage of about 1-20mg/kg of antibody may be administered, e.g., one or more administrations or continuous infusion. The antibodies of the invention may be administered in combination with other therapeutic agents, particularly with antibodies capable of neutralizing receptors involved in tumor growth or angiogenesis. Any method of administration may be used for administering the antibody of the present invention, and in particular, for example, administration may be oral, intravenous, intraperitoneal, subcutaneous, or intramuscular. The antibodies of the invention may be administered as conjugates that specifically bind to the receptor and release the toxic substance. In particular embodiments, the pharmaceutical compositions of the present invention may be administered in a single dosage form (e.g., tablets, capsules, pills, etc.). For pharmaceutical use, the compositions may be in the form of solutions, e.g., injectable solutions, emulsions, suspensions, and the like. The carrier may be any pharmaceutically suitable carrier. Preferably, the carrier used enhances the entry of the molecule into the target cell. In the pharmaceutical compositions of the invention, the antibodies may be conjugated to other therapeutic agents, such as antagonists of other growth factor receptors associated with tumorigenesis or angiogenesis, e.g., VEGFR-2, EGFR, PDGFR, receptor kinase inhibitors, BRAF inhibitors, MEK inhibitors, immunomodulatory antibodies, anti-cancer drugs such as: bevacizumab, ramucizumab, afidopril, sunitinib, pazopanib, sorafenib, cabozantinib, axitinib, regorafenib, nintedanib, lenvatinib, vemurafenib, dabrafenib, trametinib, chemotherapeutic agents, such as methylating agents (temozolomide, dacarbazine), platinum compounds (cisplatin, carboplatin, sariplatin), taxanes (taxane (paclitaxel), albumin bound taxanes, docetaxel), fluorinated pyrimidines (5-fluorouracil, capecitabine), topoisomerase I inhibitors (irinotecan, topotecan), poly (ADP-ribosidase) polymerase inhibitors (PARP) (e.g., olaparib), and the like. The pharmaceutical composition is selected according to the therapeutic requirements. These pharmaceutical compositions of the present invention can be administered in the form of tablets, capsules, oral preparations, powders, granules, tablets, liquid solutions for injection or infusion, suspensions, suppositories, inhalation preparations. See the following book for formulations: remington ("Remington: The Science and practice of medicine"), Lippincott Williams & Wilkins, 2000. One skilled in the art can select the form of administration, effective dosage, by screening for suitable diluents, adjuvants and/or excipients. The term "pharmaceutical composition" refers to such forms of formulations: the active ingredient contained therein is biologically active and free of other ingredients having unacceptable toxicity to the individual to whom the formulation is to be administered. Another aspect of the invention is a method of producing the above antibody or a synthetic or recombinant fragment thereof, comprising the steps of: culturing the host cell, and purifying the antibody or synthetic or recombinant fragment thereof from the cell culture.

In the context of the present invention, the term "comprising" also includes the term "having predominantly" or "consisting essentially of … …". In the present invention, "protein" as used herein also includes proteins, functional variants, functional derivatives, functional fragments or analogs thereof, isoforms, splice variants thereof encoded by the corresponding orthologous or homologous genes. In the present invention, "functional" means, for example, "retaining its activity". As used herein, a "fragment" refers to a polypeptide having a length of preferably at least 10 amino acids, more preferably at least 15, at least 17 or at least 20 amino acids, even more preferably at least 25 amino acids or at least 37 or 40 amino acids, and more preferably at least 50, or 100, or 150 or 200 or 250 or 300 or 350 or 400 or 450 or 500 amino acids. The "MSF fragment" disclosed herein preferably comprises the sequence VSIPPRNLGY [ aa.648 to aa.657 of SEQ ID NO: 4) (SEQ ID NO:11) ] or fragments thereof.

The invention is illustrated by reference to the non-limiting examples of the following figures.

FIG. 1 comparison of MSF and fibronectin domain structures. MSF is a truncated isoform of hFn1 produced from a single copy of fibronectin by the read-through mechanism. The 70kDa protein is identical to the amino acid sequence encoded by hFn 1N-terminal up to exon III-1a, and a unique 10 amino acid long peptide (VSIPPRNLGY (SEQ ID NO:11)) is added [ SEQ ID No. [ 4 (from aa.648 to aa.657), NCBI accession No. N.CAH60958.1 ]). [20] Figure of post-adaptation. Functional domains in Fn1 and MSF: hip1/Fib1 heparin and fibronectin binding domains; Gel-BD binding domain to gelatin/collagen; cell-BD: RGD-mediated binding to integrin; hep 2: a high affinity heparin-binding domain; fib 2: a C-terminal fibronectin binding domain; IGD motif: the isoleucine-glycine-asparagine tripeptide motif mediates prokinetic activity.

FIG. 2: exemplary description of expression vectors for transfection of CHO cells and expression of recombinant human MSF. The complete cDNA sequence of human MSF was subcloned into BamIII restriction sites present in the vector.

FIG. 3: indirect ELISA on conditioned medium from CHO-3E6 clone expressing rhMSF. Plastic wells were coated with different dilutions of conditioned medium from CHO-3E6 cell (rhMSF expressing) clones and bound MSF revealed with rabbit pAb.

FIG. 4: sera from immunized mice were titrated. Sera from immunized mice were collected after several rounds of immunization and analyzed by ELISA. Serum at various dilutions was added to multi-well plates coated with PBS, human fibronectin 1(hFn1) and His-MSF. After incubation with anti-mouse secondary antibody and addition of chromogenic substrate, the absorbance at 450nm was recorded. The reported absorbance values refer to the average obtained in a plurality of wells.

FIG. 5: the indirect ELISA was used to screen hybridoma conditioned medium for the first time. Conditioned media from hybridomas cultured in 96-well plates were evaluated for the ability to recognize human MSF by ELISA. Briefly, 50 microliters of each medium was added to multi-well plates coated with MSFVSIPPRNLGY (SEQ ID NO:11) (aa648 to aa.657 of SEQ ID NO: 4) unique decapeptides, or conditioned medium from CHO-3E6 cells expressing rhMSF, as shown. After incubation with the appropriate secondary antibody and addition of the chromogenic substrate, the absorbance at 450nm was recorded. The absorbance values reported relate to a single well.

FIG. 6: and (3) carrying out secondary screening on the hybridoma conditioned medium by indirect ELISA. Selected hybridomas (from the first screen, see fig. 5) were subcloned and cultured in 96-well cell culture plates. The conditioned medium was then tested by indirect ELISA to detect antibodies that recognize human MSF. Briefly, 50 microliters of hybridoma culture medium supernatant was added to multi-well plates coated with MSF VSIPPRNLGY (SEQ ID NO:11) (aa648 to aa.657 of SEQ ID NO: 4) unique decapeptides, or conditioned medium as described from CHO-3E6 cells expressing rhMSF, as shown. After incubation with the appropriate secondary antibody and addition of the chromogenic substrate, the absorbance at 450nm was recorded as depicted in FIG. 5. Negative controls (buffer) in wells 1A and 1B (anti-mouse IgG) and 1E and 1F (anti-rabbit IgG); positive controls (peptide or supernatant from CHO-3E 6) were placed in wells 1C and 1D (rabbit pAb).

FIG. 7: after subcloning at 0.5 cells/well, 14 clones were screened (selected from the secondary screen). 96-well plates were coated with concentrated conditioned medium (50. mu.l/well) from CHO-3ES expressing rhMSF or human hFn1 (1. mu.g/well). Each clone was tested in duplicate wells and the data are expressed as mean OD ± SD determined at 450 nm.

FIG. 8: the ELISA titrated the 1G5.3 monoclonal antibody. Microtiter plate wells were coated with human rhMSF or hFn1 (0.5. mu.g/ml) and incubated in duplicate wells with different dilutions of Ig5.3 (from 10. mu.g/ml to 0.005 ng/ml). Data are expressed as OD (mean. + -. SD) measured at 450 nm.

FIG. 9: IAC purification of recombinant human MSF. The chromatogram is reported as the UV absorbance at 280 nm. The IAC component was run on a 10% gel under denaturing and reducing conditions (input, flow through, wash, elution). Representative silver stained gels are shown in the inset (13. mu.l/lane).

FIG. 10: human cancer tissue immunostaining with the 1G5.3 antibody. Formaldehyde-fixed and paraffin-embedded human breast and lung cancer tissue sections were stained with 1G 5.3. Arrows indicate positive cells.

FIG. 11: immunofluorescent staining of human cancer tissue. Tissue samples from breast and lung cancer were stained with CD68 and 1G 5.3. Cells positive for both markers are indicated by arrows.

FIG. 12: sandwich ELISA with 1 g5.3mab. A) Standard curve run for immunoaffinity purified recombinant human MSF. B) MSF levels from CHO-3E6 cells (8.0 μ g/ml ± 3.1, mean ± SEM, n ═ 3).

FIG. 13: hFn1 spike effect in sandwich ELISA. 96-well plates were coated with 1G5.3mAb as described in FIG. 12. Buffer (PBS) or supernatant from CHO-3E6 was then added with or without hFn1 (100. mu.g/ml) to reveal bound MSF as described.

FIG. 14: MSF levels in human plasma. MSF levels in blood of healthy individuals and cancer patients were determined using a sandwich ELISA based on 1g5.3 mab. (× p ═ 0.048, mann-whitney test).

FIG. 15: anti-human MSF 1G5.3F (ab)'₂Of fragmentsPreparation and characterization. A) The 1G5.3 antibody was concentrated on a Vivaspin 610 kDa MWCO concentrator, pH3.50 relative to 100mM sodium citrate, buffer exchanged on a HiTrappParalling 5ml column, and then reacted with pepsin. An enlarged view of the 280nm uv absorbance (left axis and solid line, monitoring protein elution) and conductivity (right axis and dashed line, monitoring salt removal) from a representative SEC run is shown. B) The buffer exchanged material was incubated with pepsin and then applied to a HiTrap MabSelect 1ml column, equilibrated with PBS and eluted with 100mM sodium citrate (pH 3.50). Typical chromatograms are reported (UV absorbance at 280nm, left axis and solid line; conductivity, right axis and dashed line). Unbound (flow-through) material comprising the Fc fragment is discarded, remaining to contain F (ab)'₂The eluate of the fragments is further processed. C) The eluate from B was concentrated on a Vivaspin 610 kDa MWCO concentrator, chromatographed on a Superdex 20010/300GL column equilibrated with PBS and eluted with PBS. Protein separation (F (ab) 'monitored by UV absorbance at 280 nm'₂Left axis and solid line). Aliquots of 100 μ G of intact untreated 1G5.3 antibody were run under the same conditions (intact IgG, right axis and dashed line). Showing the overlap of the two species chromatographies. D) Aliquots of the intact 1G5.3 antibody and the corresponding F (ab)'2 fragment (from SEC in C) were separated in NuPAGE Novex Bis-Tris 10% gel under denaturing conditions in the presence or absence of dithiothreitol (+ DTT and-DTT, respectively). Coomassie blue stained gels are shown. Unreduced intact 1G5.3 and F (ab)'₂Migration to 150 and 110kDa, respectively; 1G5.3 under reducing conditions divided into two bands of 60 (heavy chain) and 25 (light chain) kDa, and F (ab)'₂One band is shown at 25 kDa. Data in a to D represent three independent experiments.

FIG. 16: comparison of background signals generated with 1G5.3mAb and 1G5.3-F (ab)'2 fragment. Buffer was added to wells coated with 1G5.3 or 1G5.3-F (ab)'2 antibody and developed as described herein. (x p ═ 0.022 student's t test).

FIG. 17: standard curves obtained were coated with 1G5.3-F (ab)'2 fragments. A) 0.2-200ng/ml hrMSF was incubated in wells coated with 1G5.3-F (ab)' 2. An overlay of 8 standard curves from a number of independent experiments is shown. B) Reported are the standard curves obtained from the average of 8 independent curves from section a). The upper and lower limits of the dynamic linear range (suitable for MSF quantitation) were 0.4 and 25ng/ml, respectively.

FIG. 18: MSF levels in healthy individuals and PDAC patients. MSF was determined by sandwich ELISA using 1G5.3-F (ab)'2 as capture antibody. A) Distribution of MSF levels in healthy individuals (n-28). B) The MSF levels in PDAC patients (n-33) were compared to those in healthy individuals. (. P <0.0001, mann-whitney test).

FIG. 19: dot blot analysis of mabs 7.1 and 1G5.3 interaction with rhMSF. Recombinant human MSF (rhMSF) was adsorbed onto nitrocellulose membrane (200 ng/well) using a Bio-Dot apparatus (Bio-Rad). After blocking the uncoated sites, spots were probed with conditioned medium (undiluted supernatant) from mAb7.1 hybridoma cultures or purified 1G5.3 monoclonal antibody (250 ng/ml). Blank wells (without rhMSF) were used as negative controls (Ctrl). One blot is shown representing the spots of two independent experiments.

FIG. 20: indirect ELISA analysis of mabs 7.1 and 1G5.3 interaction with biot-VSIPPRNLGY and rhMSF. A) The biot-VSIPPRNLGY peptide (SEQ ID NO:11) (aa648-657 sequence spanning the C-terminus of human MSF) was captured on a Maxisorb plate coated with NeutrAvidin. Conditioned media from mab7.1 hybridoma culture (undiluted) or purified 1G5.3(10ng/ml) was added and bound antibodies were revealed with appropriate HRP-conjugated secondary antibodies as described in the materials and methods section. B) Maxisorb plates were coated with rhMSF and incubated with mab7.1 hybridoma cultures or purified 1G 5.3. Binding of the antibody was revealed as described in A. The results in both A and B are expressed as absorbance at 450nm (A450nm, mean. + -. SD). Wells containing NeutrAvidin (NeutrAvidin in a) or PBS-/- (PBS in B) alone served as negative controls. A graph of one of two independent experiments, each run in duplicate, is shown.

FIG. 21: dose-dependence of the interaction of mAb7.1 and 1G5.3 with biolt-VSIPPRNLGY (SEQ ID NO:11) and rhMSF. A) The biot-VSIPPRNLGY peptide (SEQ ID No:11) was captured on a Maxisorb plate coated with NeutrAcidin, as shown in FIG. 20. Conditioned media from mab7.1 or 1G5.3 hybridoma cell cultures were added at the indicated concentrations. Conjugated secondary antibodies with appropriate HRP revealed bound antibodies as described in materials and methods. Maxisorb plates were coated with rhMSF and incubated with conditioned media from mab7.1 or 1G5.3 hybridoma cell cultures at the indicated dilutions. Binding of the antibody was revealed as indicated at a. Results in both A and B are expressed as absorbance at 450nm (A450nm, mean. + -. SD) after subtraction of background signal from blank wells (i.e. containing NeutrAvidin or PBS-/-). A graph of one of two independent experiments, each run in duplicate, is shown.

Example 1

Materials and methods

Commercially available reagents and cell lines

Human fibronectin 1(hFn1) was obtained from Calbiochem (Merck, Milan, Italy; catalog number 341635). Low endotoxin Fetal Calf Serum (FCS) was purchased from Sigma Aldrich (Sigma Aldrich) (Milan, Italy; Cat. No: F7524); RPMI-1640, modified Du's Medium (DMEM) and trypsin used for cell culture were from LONZA (Euroclone, Milan Italy; catalog Nos. BE 12-167F; BE12-733F and BE17-161E, respectively). Phosphate Buffered Saline (PBS) with calcium and magnesium (PBS +/+) from Biosera (Biotecna, Milan, Italy; Cat. No.: XC-S2067), calcium and magnesium free PBS (PBS-/-Cat. No.: D8537) and geneticin (G418; Cat. No.: G8168) from Sigma Aldrich Corp.

Synthetic peptides specific for human MSF (aa648-657 of SEQ ID No: 4) were synthesized by PRIMM S.r.l. (Milan, Italy Milan). A cysteine residue was added to the COOH-terminus of a 10 amino acid long peptide (VSIPPRNLGYC [ SEQ ID NO:9 ]). The peptide was conjugated to Keyhole Limpet Hemocyanin (KLH) (VSIPPRNLGYC-KLH) (SEQ ID NO: 9).

A105 amino acid fragment of human MSF (His-MSF; residues 553 to 657 of the C-terminal part of MSF; molecular weight 14.335kDa, SEQ ID NO:10), including a C-terminal specific MSF decapeptide VSIPPRNLGY (SEQ ID NO:11) and an N-terminal histidine tag, was obtained from PRIMM. Expression vector pSG5(4,100bp) was from Stratagene (La Jolla, Calif.); pSV2neo, used to confer resistance to selectable marker G418, was obtained from ATCC (Manassas, Va.). By using2000 (Invitrogen) CHO cells. CHO (Chinese hamster ovary) cells and SP2/0 myeloma cells were obtained from ATCC (catalog numbers ATCC CCL-61 and CRL 1581, respectively) and cultured in Dutch modified Israh's medium (DMEM) supplemented with L-glutamine (Longsa, catalog numbers: BE17-605E/U1) and 10% (v/v) FCS.

Expression of human recombinant MSF

Recombinant human msf (rhmsf) was expressed in CHO cells. The full-length cDNA (2,192bp, accession AJ535086.1, SEQ ID No:3) of human MSF was subcloned into the BamH1 site of pSG5 (FIG. 2, pSG-MSF). The orientation of the cloned MSF was determined by sequencing. CHO cells were co-transfected with the pSV2neo vector according to the manufacturer's protocol, conferring G418 resistance. Transfected clones were screened at 800. mu.g/ml G418 and assayed for MSF production by indirect ELISA (see below) using rabbit polyclonal antisera (generated by inoculation with MSF decapeptide in rabbits). CHO-3E6 cells were obtained by further subcloning one of the positive clones by limiting dilution, which produced high levels of human recombinant MSF.

Production and purification of antibodies against human recombinant MSF

Rabbit polyclonal antiserum (pAb) was generated by immunizing rabbits with a synthetic 10 amino acid-long peptide specific for human MSF, to which a cysteine (SEQ ID NO:9) was added at the COOH terminus, which was conjugated to KLH as a carrier (VSIPPRNLGYC-KLH) (SEQ ID NO: 9). Rabbits were challenged intraperitoneally with 300 μ g of MSF-specific peptide diluted in complete freund's adjuvant. Immunizations were repeated at days 21, 28 and 35 with peptides diluted in incomplete freund's adjuvant. Blood from immunized rabbits (40-50 ml/rabbit) was collected, tested by ELISA against the immunogen (VSIPPRNLGYC-KLH) (SEQ ID NO:9), and specific antibodies were purified by immunoaffinity on CNBr-agarose columns carrying the same immunogen.

Balb/c mice (BALB/cAnNCrl, Charles River, Italy, Carl family (Calco)) were immunized to obtain monoclonal antibodies against rhMSF. Briefly, 200. mu.l of supernatant from CHO-3E6 cells expressing rhMSF were separated on a 10% polyacrylamide gel under reducing and denaturing conditions in a discontinuous buffer system of Laemmli. Colloidal coomassie for gelSi (Bio-Safe)^TMCoomassie, Cat # 161-0786, Burley (Bio-Rad)) stained, the 70kDa MSF band excised and fragmented in PBS. Balb/c mice, 8 weeks old, were immunized with the suspension. The procedure was repeated three times with 3 week intervals. A fourth challenge was performed with purified His-MSF (20. mu.g/mouse). Antibody titers were analyzed by indirect ELISA against His-MSF and Fn1 (used as negative control, see below). Splenocytes from responding mice were fused with SP2/0 myeloma using polyethylene Glycon1550(SERVA, italy roman) according to the standard procedure of the manufacturer. Cells were seeded in 96-well plates and screened with HAT medium (RPMI-1640 medium containing 10% FCS,100mg/mL streptomycin, 100IU/mL penicillin, 100mM hypoxanthine, 16mM thymidine, and 400mM aminopterin). After 2 weeks, the culture supernatants were screened for antibody reactivity and specificity using an indirect ELISA against the peptide, using the supernatant of CHO-3E6 as a source of human recombinant MSF. Cells from 4 different positive IgG production wells were subcloned at 5 cells/well (two 96-well plates per clone). A second screen was performed with indirect ELISA against MSF decapeptide and rhMSF contained in CHO-3E6 cell supernatant. Cells from two different IgG production wells were again subcloned at 0.5 cells/well. This subcloned Ig hybridoma was tested by indirect ELISA against rhMSF and Fn1 contained in CHO-3E6 cell supernatant. A total of 14 hybridomas were found to recognize rhMSF contained in CHO-3E6 culture supernatant, but not Fn 1. Hybridoma 1G5.3 was selected for further development. Monoclonal antibodies secreted from the 1G5.3 hybridoma, now designated 1G5.3, were purified from a protein G-Sepharose 4Fast Flow column (GE healthcare, Pittsburgh, Pa., Cat. No.: 17061801) according to the manufacturer's instructions. Briefly, protein G-Sepharose columns (1ml column volume) were equilibrated with PBS +/+ and culture supernatants diluted with the same buffer were loaded. Flow-through was again applied to the column and the process was repeated 3 times. The MSF-specific antibodies were then eluted with 0.1M glycine-HCl (pH2.8) and immediately buffered with 1.5M Tris-HCl (pH 8.8). The isotype of 1G5.3 was determined using the mouse monoclonal antibody isotype test kit (Berle, Cat: MMT 1). Rabbit mAb RM103 (Abcam, Cat.: 190484; anti-mouse kappa light chain) and rat mAb JC5-1 (Abcamu, Cat.: 99622, anti-mouse lambda) were usedLight chain) the light chain type was determined by Western blot.

Determination of 1G5.3 sequence

Use ofReagents (Ambion, Cat.: 15596-. Total RNA is then reverse transcribed into cDNA using an isotype-specific antisense primer or a universal primer according to the technical manual of the PrimeScript first strand cDNA Synthesis kit (Takara, Cat. No.: 6110A). Antibody fragments of VH, VL, CH and CL were amplified according to the Standard Operating Procedure (SOP) of Rapid Amplification of CDNA Ends (RACE) of GenScript. The amplified antibody fragments were cloned into standard cloning vectors, respectively. Colony PCR was performed to screen clones with inserts of the correct size. For each fragment, no less than five clones with the correct size insert were sequenced. The different cloned sequences are aligned to provide a consensus sequence.

F(ab)’₂Preparation of fragments

From 1G5.3[1G5.3-F (ab)'₂]Production of F (ab) 'by enzyme treatment'₂And (3) fragment. An aliquot of 8ml (8mg) of the 1G5.3 monoclonal antibody (1mg/ml in PBS solution) (Sigma Aldrich, Cat. No: D1408) was concentrated to 1ml on a Vivaspin6PES 10kDa MWCO concentrator (Sartorius Stedim, Germany, Gottingen (Goettingen), Cat. No: VS0602), 100mM sodium citrate (pH3.50) (Merck Millipore, Germany, Damm Stattat (Darmstadt), Cat. No. 17-1408-01) was buffer exchanged twice (500. mu.l/time) in succession on a HiTrap desalting 5ml column (GE healthcare Co., Cat. No: 17-1408-01). Protein containing fractions (total volume 4ml) were pooled and antibody concentration determined by UV absorbance at 280nm using a value of 1.4 as the extinction coefficient of mouse IgG1 at 280nm (expressed as absorbance of 0.1% (w/v) solution at 280 nm).

To produce 1G5.3-F (ab)'₂Fragments, 5. mu.g pepsin (Sigma Aldrich, Cat. No: P6887) per mg antibody, and the resulting mixture incubated at 37 ℃ for 16 hThen (c) is performed. The reaction was blocked by adjusting the pH of the solution to 7.0 by adding 650. mu.l of 1M Tris-Cl, pH 8.80 (Merck Michiobo, Cat. No.: 108382). The solution was then applied to a HiTrapDiabSelect protein A1ml column (GE healthcare), equilibrated with PBS and eluted with 100mM sodium citrate (pH3.5) at 1 ml/min. Fractions containing F (ab)'2 (4 ml total volume) were pooled and concentrated to 600. mu.l with a Vivaspin6PES 10kDa MWCO concentrator. The concentrated material was chromatographed on a Superdex 20010/300GL column (GE healthcare), equilibrated with PBS and eluted at 0.5 ml/min in PBS. F (ab) 'of the eluted fractions was determined as described above'₂Fragment, SEC purified material stored at-20 ℃ until use. All chromatographies are carried out inOn the Purifier FPLC System (GE healthcare); protein elution and salt separation were monitored by ultraviolet absorbance at 280nm and conductivity (mS/cm), respectively. A3 μ G aliquot of intact 1G5.3 antibody and corresponding 1G5.3-F (ab)'₂Fragments (from SEC) were isolated in NuPAGE Novex Bis-Tris 10% gel (Seimer Feishell Scientific, Waltham, Mass.) under denaturing conditions in the presence or absence of dithiothreitol. After electrophoresis, proteins were detected using Bio-Safe Coomassie dye (Burley, Hercules, Calif.).

Purification of human recombinant MSF

Human recombinant MSF was purified from conditioned medium of CHO-3E6 by immunoaffinity chromatography (IAC). 400ml of conditioned medium was added at 2.5 ml/minA5 ml HiTrap NHS-activated HP column (GE healthcare Co., Ltd., Pittsburgh, Pa.) covalently coupled with 1G5.3(2.8mg 1G5.3/ml affinity medium) in the Purifier FPLC System (GE healthcare Co., Ltd.) was equilibrated with buffer A (50mM Tris-HCl,150mM NaCl, pH 7.00). The 1G5.3 column was washed extensively with buffer A, then buffer B (50mM Tris-HCl,500mM NaCl, pH7.00) at 5 ml/min, and bound rhMSF was eluted with buffer C (3.5M MgCl2) in a total volume of 4 ml. By usingAnalytical grade Superdex 20010/300GL gel filtration System (GE healthcare) on the Purifier FPLC System to assess homogeneity of the eluted proteins, equilibrated and eluted at 0.5 ml/min using buffer B. In addition, aliquots of conditioned medium (input), unbound material (flow-through) and eluted protein (elution) from the IAC were resolved on NuPAGE Novex Bis-Tris 10% gel (seimer fisher science, waltham, massachusetts) under denaturing and reducing conditions (i.e., in the presence of dithiothreitol). After electrophoresis, the proteins were resolved using ProteoSilver Plus silver stain kit (sigma aldrich, st. louis) and Bio-Safe coomassie dye (berle, helaklesch, ca) or transferred to polyvinylidene fluoride (PVDF) membranes for subsequent immunodetection using anti-human MSF or anti-hFn 1 rabbit polyclonal antibodies, followed by fully donkey antibody (GE healthcare) conjugated anti-rabbit IgG horseradish peroxidase (HRP). The membrane was developed with Immobilon western HRP substrate (Merck Miclo, Germany, Dammstat) and the chemiluminescence recorded on a ChemicosmP system (Berle). Throughout the purification process, the total protein content was determined by the Bradford protein assay (burle) and rhMSF-specific titers were determined by the "internal" ELISA MSF assay described below.

Immunohistochemical analysis of human tumor sections

Paraffin-embedded human tissue sections were excised and stored overnight at 37 ℃. Sections were deparaffinized in a bioclean. Antigen unmasking was performed in a Decoloker chamber in DIVA buffer 1X (catalog number DV2004 of Biomedicine, Inc.: Biocare Medical), at 125 ℃ for 3 minutes and at 90 ℃ for 5 minutes. Endogenous peroxidase was blocked with Peroxidized-1 (Bio-medical Co., Cat: PX968) for 15 minutes, and then non-specific binding sites were blocked with Background Sniper solution (Bio-medical Co., Cat: BS966) for 30 minutes. The human tissue samples were then incubated with 1G5.3(1/150-1/300) for 1 hour to identify MSF, or macrophages were identified with a mouse monoclonal anti-human CD68 (Dako catalog # MO876, clone PG-M1,780 μ G/ml for 1 hour). Staining with CD68 does not require antigen unmasking. Immunostaining for 1G5.3 or CD68 was revealed after 20 minutes incubation with rabbit anti-mouse MACH1 Polymer-HRP (biomedical Co., Cat: MIU 539). Then, the reaction mixture was subjected to color reaction with 3,3' -diaminobenzidine tetrahydrochloride (Bio-medical Co., Ltd., Cat: DB 801). The slides were counterstained with hematoxylin solution for 3 minutes.

Indirect ELISA

ELISA plates (Nunc Maxisorb Immunoplate Cat: 446612) were coated with His-MSF (1-0.5. mu.g/well), supernatant from CHO-3E6 (50. mu.l) or hFn1 (1. mu.g/well) (diluted with 15mM sodium carbonate buffer (pH 9.6)) (overnight at 4 ℃). After coating, the plates were washed three times with PBS +/+ and 0.05% (v/v) Tween20 (wash buffer) and incubated with 5% (w/v) skim milk (in wash buffer) for 2 hours at room temperature to block non-specific interactions. The wells were washed three times with wash buffer, then either rabbit polyclonal antiserum, supernatant from anti-MSF hybridoma or an aliquot of purified 1G5.3 diluted in wash buffer was added and incubated for 1 hour at room temperature. Anti-rabbit IgG or anti-mouse IgG (GENA 934 and GENA931, catalog numbers, respectively, GE healthcare) labeled with horseradish peroxidase was then added (1/2000, in wash buffer) and incubated at room temperature for 1 hour. With 3,3',5,5' -tetramethylbenzidine (TMB; 1 Step)^TMULTRA TMB-ELISA, Sammerfscience, Thermoscientific, Rockford, Ill., USA; catalog No. 34019) the reaction was developed, quenched with 2NH2SO4, and then the absorbance at 450nm was read with an automatic plate reader (Versamax microtiter plate reader). Each sample was analyzed in triplicate and the results reported as mean OD450 ± SD or SEM, as shown in the figure. anti-MSF antibodies were added to the buffer coated wells to obtain baseline.

Sandwich ELISA

For determination of MSF levels in biological fluids, a sandwich ELISA was developed. For this, ELISA plates were incubated overnight at 4 ℃ with 250 ng/well of 1G5.3 or 500 ng/well of 1G5.3-F (ab)'2 fragment diluted in PBS-/- (pH7.0). After coating, the plates were washed three times with 300. mu.l of wash buffer and then blocked with 5% skim milk at 37 ℃ for 2 hours. 1/10 was then added and incubated for 1 hour at room temperature in 100. mu.l of human plasma (informed consent) diluted in washing buffer containing 2% fetal bovine serum albumin. In the same plate, a standard curve was generated with purified recombinant human MSF. After washing, the plates were incubated with a commercial biotinylated polyclonal sheep IgG recognizing human hFn1 (0.25. mu.g/ml in wash buffer; catalog number R & D: BAF1918) for 1 hour at room temperature. Finally, 100. mu.l peroxidase-streptavidin (BioSpa Cat: SB01-161) diluted in washing buffer was added 1/10000 and incubated for 1 hour at room temperature. The plates were then washed, incubated with 100. mu.l/well of chromogenic substrate TMB, and then blocked with 2NH2SO 4. The absorbance at 450nm was read as described above. The concentration of MSF in plasma samples was calculated by linear regression according to a standard curve prepared with recombinant MSF.

Results

The present invention relates to the detection of human MSF as a diagnostic and prognostic marker of M2 polarized macrophages involved in inflammatory pathologies such as asthma, allergy and cancer. To this end, the inventors developed the monoclonal antibody 1G5.3 that specifically recognizes rhMSF. The antibody 1G5.3, which can be used for immunoaffinity purification of rhMSF, is effective in immunohistochemistry by ELISA. Finally and most importantly, 1g5.3mab has been used efficiently to develop specific ELISA systems for determining human MSF levels in biological fluids. This assay is based on our particularly advantageous 1G5.3 antibody, antibody fragment or derivative thereof that specifically recognizes MSF, and on the use of full-length human recombinant MSF as a standard.

Production of human recombinant MSF

Human recombinant MSF was purified from the supernatant of pSG-MSF transfected CHO cells. Approximately 300 clones derived from CHO cells transfected with pSG-MSF and pSVneo were analyzed by indirect ELISA, using initially pAb generated by immunizing rabbits with MSF-specific peptides coupled to KLH. Only 6 clones produced the desired level of rhMSF, which was further subcloned by limiting dilution. FIG. 3 reports the results of an indirect ELISA showing the titration curve of the supernatant collected from clone CHO-3E6, which was selected as a source of recombinant protein for further development.

Production and characterization of 1G5.3 mAb. As detailed in the materials and methods section, monoclonal antibody 1G5.3 was generated by immunizing mice with rhMSF. Sera from immunized mice were analyzed by indirect ELISA with His-MSF as positive control and hFn1 as negative control. As shown in fig. 4, serum recognized a 100 amino acid fragment of human MSF (His-MSF) but not hFn1 under at least the experimental setup used herein.

Splenocytes from responding mice were then fused with SP2/0 myeloma and plated in 96-well plates. A total of 5 plates were prepared. After 2 weeks, fusion was tested by indirect ELISA: 100 μ l of supernatant from each well of 5 pieces of 96-well plates was dispensed into wells previously coated with CHO-3E6 cell supernatant used as a source of recombinant MSF or with specific MSF decapeptide. Figure 5 reports the results of one plate analyzed. From this initial screen, the inventors selected four wells (G5 and B8 for plate 1, a5 and D7 for plate 5) and subcloned at 5 cells/well (2 plates per clone).

The supernatants from these plates were used for secondary screening by indirect ELISA for peptides and supernatants from CHO-3E6 cells as a source of recombinant MSF. FIG. 6 reports the screening of two plates derived from clone G5. From this screen, the inventors selected two clones, F7 of plate 1 and B8 of plate 2, subcloned at 0.5 cells/well. 10 plates were obtained and supernatants were tested on microtiter plates coated with peptides or supernatants from CHO-3E6 cells as a source of rhMSF. From this analysis, the inventors selected 14 clones, named 1G5.1, 1G5.2, 1G5.3 … … up to 1G 5.14. Clones were cultured in 6-well plates and again tested in CHO-3E6 transfected cell supernatants and hFn1 for their ability to recognize MSF. As shown in fig. 7, none of these clones identified hFn1, but MSF with varying potency. Clone 1G5.3 was selected for further development.

Mab1g5.3 was purified from hybridoma cell culture supernatants by affinity chromatography on protein-G sepharose. Hybridoma 1G5.3 expresses secreted antibodies of subtype 1G1 heavy chain and kappa light chain. The purified antibody was finally analyzed by ELISA. FIG. 8 shows the results of titration curves of purified monoclonal antibody 1G5.3 against plates coated with rhMSF or hFn1 (both 500 ng/well). The results confirm that mAb1G5.3 does not recognize human hFn 1.

1G5.3 DNA and protein sequences

After subcloning both strands, the heavy and light chain DNA sequences of 1G5.3 were obtained. The DNA sequence of the mouse heavy chain of 1G5.3 is shown as SEQ ID NO.5, and the protein sequence is shown as SEQ ID NO. 6. 1G5.3 has a light chain DNA sequence shown in SEQ ID NO.7 and a protein sequence shown in SEQ ID NO. 8.

mAb1G5.3 for purification of rhMSF

rhMSF secreted by CHO-3E6 cells in culture supernatants can be purified efficiently by immunoaffinity chromatography (IAC). Conditioned medium (input) from CHO-E6 cells was passed through a 1G5.3 column equilibrated and eluted as described in materials and methods. Typical results of the purification process are shown in FIG. 9.

mAb1G5.3 for immunohistochemical analysis

1G5.3 was effective in immunohistochemical analysis, recognizing MSF in human cancer tissues, as shown in FIG. 10. The protein is expressed in cancer cells, also in somatic cells in breast cancer tissue, and in alveolar macrophages in lung cancer tissue (as judged morphologically). Double immunofluorescent staining with the macrophage marker CD68 confirmed expression in alveolar macrophages and a portion of macrophages from breast cancer specimens in lung cancer tissues (fig. 11).

mAb1G5.3 for use in developing immunoassays for determination of MSF levels in biological fluids

To develop an immunoassay to test MSF levels in biological fluids, the inventors tested 1G5.3 as a capture antibody. To generate a standard curve, plastic wells in ELISA microtiter plates were coated with 250 ng/well mAb1G5.3 diluted in carbonate buffer. Non-specific sites were then blocked and serial dilutions of rhMSF (concentration range 1.5-1000ng/ml) were dispensed into duplicate wells. Wells coated with buffer only served as negative controls. ELISA was performed as detailed in the materials and methods section, revealing bound MSF by incubation with commercial sheep antiserum to hFn1 at the end of the process. Figure 12A reports a typical standard curve obtained according to this procedure. Initially, the inventors determined MSF levels in CHO-3E6 cell culture supernatants using this assay. As shown in FIG. 12B, the MSF concentration in the supernatant from CHO-3E6 was approximately 8.0. + -. 3.1. mu.g/ml (mean. + -. SEM; three different supernatants).

To verify this, ELISA specifically recognized human MSF, adding up to 100 μ g/ml purified hFn1 to the assay, despite using anti-hFn 1 as the detection antibody. As shown in fig. 13, hFn1 was not shown in the developed sandwich ELISA, and addition of hFn1 did not affect recognition of human MSF. The results confirm the specificity of 1G5.3 in recognizing and capturing human MSF, but not hFn 1. The inventors then tested this ELISA assay to assess MSF content in plasma samples of healthy individuals and cancer patients (informed consent was obtained). FIG. 14 reports the results obtained with the developed sandwich ELISA. MSF can be measured in plasma of both healthy individuals and cancer patients. In addition, as shown in figure 14, patients with cancer had higher levels of MSF compared to healthy individuals (p ═ 0.048, mann-whitney test).

1G5.3-F(ab)’₂Generation of fragments

Sandwich ELISA based on the use of our mAb1G5.3 was effective in determining MSF levels in culture supernatants and plasma samples. To improve the specificity of ELISA, the inventors also tested 1G5.3-F (ab)'2 obtained after treating purified antibody with pepsin. For this purpose, the 1G5.3 antibody was concentrated on a Vivaspin6 concentrator (10kDa MWCO), the buffer was exchanged on a HiTrap Desainting 5ml column, relative to 100mM sodium citrate pH3.50, and then reacted with pepsin. FIG. 15A shows a typical profile after protein separation by SEC, before pepsin treatment. The buffer exchanged material was incubated with pepsin (37 ℃ for 14 hours) and then applied to a HiTrap MabSelect column to isolate the Fc fragment, and unbound material was recovered from the f (ab)'2 fragment (flow through) and eluted from the column with 100mM sodium citrate (fig. 15B). The F (ab)'2 fragment eluted from the column was then concentrated with Vivaspin6, chromatographed on a Superdex 20010/300GL column, equilibrated and eluted with PBS (FIG. 16C). Aliquots of 100 μ G of intact untreated 1G5.3 antibody were run under the same conditions (intact IgG, right axis and dashed line, fig. 15C). Aliquots of the intact 1G5.3 antibody and the corresponding F (ab)'2 fragment (from SEC in C) were separated in NuPAGE Novex Bis-Tris 10% gel under denaturing conditions in the presence or absence of dithiothreitol (+ DTT and-DTT, respectively). Figure 15D reports a representative gel stained with coomassie. Unreduced 1G5.3 and 1G5.3F (ab)'₂Migration to 150 and 110kDa, respectively; 1G5.3 min under reducing conditionsTwo bands of 60 (heavy chain) and 25 (light chain) kDa, and 1G5.3-F (ab)'₂One band is shown at 25 kDa.

1G5.3-F (ab)'2 fragments for use in the development of immunoassays for determining MSF levels in biological fluids

The 1G5.3-F (ab)'2 fragment was then tested in a sandwich ELISA according to the same procedure described above. The inventors initially compared the background obtained with two different antibodies coated in plastic wells. As shown in fig. 16, the background was significantly reduced by coating with 1G5.3-f (ab) '2 (p ═ 0.022; student's t test). This is important to increase the lower limit of detection of the assay. To determine the optimal range of rhMSF concentrations for the standard curve, different amounts of protein (0.2-200ng/ml) were incubated on 1G5.3-F (ab)'2 coated plates. The optimal range result for the rhMSF standard curve is 0.4-25mg/ml, which results in an improvement in assay sensitivity over a 1G5.3mAb (lower detection limit of 1.5mg/ml) based setting. Fig. 17A shows the reproducibility of the obtained standard curve, and fig. 17B shows the generated standard curve. The inventors then determined MSF levels in a series of plasma collected from healthy donors and patients with Pancreatic Ductal Adenocarcinoma (PDAC) who had obtained informed consent. Median MSF levels were 11.11ng/ml for healthy donors (Q1-Q3: 4.64-17.97; n-28; fig. 18A), 143.7ng/ml in PDAC patients (Q1-Q3:111,6-176, 8; n-33; fig. 18B), with statistically significant differences from levels of healthy individuals (p <0.0001 man-whitney test). In summary, the data presented herein suggest that 1G5.3 is a specific monoclonal antibody that recognizes MSF, a molecule associated with M2 polarized macrophages. Antibodies and derivatives thereof are highly effective in quantifying MSF levels in a biological fluid of an individual having a pathological state associated with M2 polarization of macrophages.

Clinical application

The developed monoclonal antibodies can be used to identify M2 polarized macrophages (involved in different inflammatory conditions) and M2-like tumor-bound macrophages. In addition, the antibodies can be used to develop assays for assessing circulating levels of proteins. Analysis of MSF expression can have diagnostic and prognostic value in cancer patients and others.

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Anti-human Migration Stimulating Factor (MSF) and uses thereof

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